This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Introduction The influenza A virus is a significant concern of public health. It accounts for 36,000 deaths, 200,000 hospitalizations per year resulting in $10 billion in health care costs in the US. Although remarkable advances have been achieved in medicine in the past century, many questions remain regarding infection with influenza. One aspect of influenza infection is the inhibition of the activity of viral protein hemagglutinin (HA) by binding proteins such as lung surfactant proteins (SP-D or A). Although a strong consensus in the literature concerning the interaction of HA and SP involves their N-glycosylation sites, limited structural information concerning the glycans have been reported. We explored a comprehensive mass spectrometry strategy to study HA and SP-D in terms of identification of N-glycosylation sites and the carbohydrate structures in order to meet the need for a detailed understanding of influenza pathobiology. Methods HA from H3N2 strain Philippines 1982 and the truncated pig SP-D protein containing the N-glycosylation site of interest were studied. Trypsin digestion was conducted at 37[unreadable]C overnight with 1:50 enzyme-protein (w/w) ratio in 50mM Hepes buffer (pH 8) and desalted with micro-reversed phase chromatography. A PNGase-F digestion was then conducted overnight in water or heavy water to allow for relative quantification of N-linked sites. All samples were analyzed by LC-normal phase-MS/MS in positive ion mode on an LTQ-Orbitrap XL. In order to determine the maximum structural information on the carbohydrates and parent peptides, the glycosyled peptides were identified using the Glycomod program with confirmation using manual interpretation of tandem MS data. Premilary results From the comparison of the LC-MS data obtained from the enzymatic deglycosylation in O18 water, the number, the specific location and the relative site occupancy of glycosylation were clearly identified. In total, 5 of the 13 theoretical N-glycosylation sites were confirmed in HA, and a single site was observed for pig SP-D truncated protein. Site occupancy of 100% was observed for almost all of the sites. The high resolution (60,000) and the mass accuracy (less than one 2ppm) provided by the high resolution Orbitrap mass spectrometer was essential to remove ambiguities concerning the identification of the site and the sequence of the peptides involved. Depending on the glycosylated site, different carbohydrate structures were observed, including high mannose and complex N-glycans on HA. At this point of our interpretation, sulfation and the presence of sialic acid have not been identified for H3N2 HA. Hybrid complex carbohydrates were clearly demonstrated to be present on the site of glycosylation of the pig SP-D protein. The complementarily nature of different modes of fragmentation (ETD, CID, HCD) have been exploited to collect enhanced structural information of the glycopeptides given the complexity of the data. An example for illustrating our results is the glycosylation of site N165 of HA. The MS and MS/MS data indicated the presence of N-glycans at this site as predominantly composed of high mannose carbohydrates: Hex2to 7 -Man3GlcNAc2. Harshtorm et al (2008) and Hartley et al (1997) support the implication of high mannoses carnohydrates of N165 in the inhibition of the HA activity.